This invention relates to a reagent for quantitating red blood cells, particularly mature erythrocytes and reticulocytes, adapted to use in the optical measurement of blood through flow cytometry.
Immature erythrocytes in a blood specimen are referred to as reticulocytes and ordinarily make up 0.7 to 2.2% of the total red blood cell population. Reticulocytes are of vital importance in modern clinical tests as their quantitative determination provides support in diagnosing acute internal hemorrhage, hemolytic anemia and aplastic anemia.
Smears stained with a basic dye such as new methylene blue or brilliant cresyl blue are used in quantitating reticulocytes as a percentage of total red blood cell content based on observation of the dyed reticulocytes under a microscope. However, staining with a dye of the aforementioned type requires considerable time and labor with regard to pretreatment of the blood specimen and visual enumeration of the reticulocytes after they are stained. For these reasons, such method is unsuitable for cases where a large number of tests are to be performed. Accordingly, a blood test using flow cytometry has been developed and is finding success in raising the speed and accuracy of measurement. However, various problems still remain as far as the quantitating of reticulocytes is concerned.
Examples of the prior art include the teachings of U.S. Pat. Nos. 4,336,029, 4,325,706 and 4,284,412, which propose the use of acridine orange for staining reticulocytes. Acridine orange produces red fluorescence upon being adsorbed by the reticulocyte RNA component and makes quantitative determination of the reticulocytes possible by allowing the red fluorescence to be distinguished from the green fluorescence produced by mature erythrocytes, which constitute the majority of the red blood cell population. However, the use of acridine orange is accompanied by the following problems: (A) The dye solution itself exhibits strong background fluorescence, which appears as background noise when measuring the intensity of fluorescence originating from the blood cells. The result is the likelihood of measurement error. (B) Since the fluorescence is beyond the red region of 630 nm, a highly sensitive fluorometric apparatus is required. (C) Since non-specific staining proceeds to be high degree and red fluorescence is produced in the mature erythrocytes, it is difficult to prepare a dye composition with little error from one blood sample to another. (D) The platelets are stained strongly and both the platelets and red blood cells are passed through a counting aperture in the flow cytometer simultaneously during measurement. When this is done, the intensity of light scattered by the mature erythrocytes and the intensity of red fluorescence from the platelets are measured at the same time. This constitutes the reticulocyte signal level and, hence, is a source of error. (E) The acridine orange solution is strongly dependent upon the environment, so that the intensity of fluorescence tends to vary.
The specification of Japanese Patent Application Laid-Open No. 59-142465 discloses use of thioflavine T as the dye. Following RNA staining a blood specimen using the proposed dye, background fluorescence and non-specific fluorescence of mature red blood cells are reduced by dilution and washing, after which reticulocyte RNA fluorescence is sensed and measured by a flow cytometer. The disadvantages of this approach are as follows: (F) Specific fluorescence caused by the dye for RNA bonding is also reduced with the reduction in non-specific fluorescence through dilution. This demands use of photometric equipment which is highly sensitive to fluorescence. (G) Since the decline in the intensity of fluorescence with time is quite sudden after dilution, data having a high degree of reproducibility cannot be obtained. (G') Dyeing requires a comparatively long period of time.
Pyronine Y is also used as a dye. Fixed red blood cells are washed after RNA staining to completely remove nonspecific fluorescence and background fluorescence, after which specific fluorescence caused by the dye for RNA bonding is measured. Here the drawbacks are: (H) The process steps of fixing the blood cells in advance, staining and washing are time consuming and require much more time than other methods. As a result, this approach is not suitable for dealing with a large number of samples. (1) Pyronine Y itself has a very low fluorescence quantum efficiency and, hence, measurement is possible only with excitation using a laser source having a large output. (J) Since pyronine Y will form bonds only with RNA and DNA of a low polymerization degree, the efficiency of RNA staining for obtaining specific fluorescence is poor.